1. Field of the Invention
The present invention relates generally to the fields of cellular and molecular biology. More particularly, it concerns the use of peptides that targets tumor tissue and describes methods to achieve targeted delivery of anticancer drugs using this peptide. The invention also contemplates methods for using the peptide for the diagnosis and imaging of cancer.
2. Description of Related Art
A. Cancers
Cancer is one of the leading causes of disease, being responsible for 526,000 deaths in the United States each year. In 1998, the American Cancer Society estimated that 60,000 Americans would be diagnosed with head and neck cancer. Head and neck cancer is the term given to a variety of malignant tumors that may occur in the head and neck region: the oral cavity (including the tissues of the lip or mouth such as the tongue, the gums, the lining of the cheeks and lips, the bottom of the mouth, the hard and soft palate and the retromolar trigone); the pharynx (including the hypopharynx, nasopharynx and oropharynx, also called the throat); paranasal sinuses (including the frontal sinuses above the nose, the maxillary sinuses in the upper part of either side of the upper jawbone, the ethmoid sinuses just behind either side of the upper nose, and the sphenoid sinus behind the ethmoid sinus in the center of the skull) and nasal cavity; the larynx (also called the voicebox); thyroid gland (including cancers of the thyroid which are papillary, follicular, medullary and anaplastic); parathyroid gland; salivary glands (including the major clusters of salivary glands found below the tongue, on the sides of the face just in front of the ears, and under the jawbone); lesions of the skin of the face and neck and the cervical lymph nodes; and metastatic squamous neck cancer with occult primary.
Although the percentage of oral and head and neck cancer patients in the United States is only about 5% of all cancers diagnosed, the importance of this disease is heightened by the fact that functional and aesthetic problems are commonly associated with this type of cancer and its treatment. Estimates indicate that there are more than 500,000 survivors of oral and head and neck cancer living in the United States today. Coping with this type of cancer can be extremely difficult. Not only can the disease be life-threatening, but many patients must also endure alterations in facial and neck appearance, as well as alterations in speech, sight, smell, chewing, swallowing and taste perception.
Head and neck cancers can arise from squamous cell carcinomas (SCC), which are the second most common form of skin cancer. They occur in men more often than women and originate primarily in skin exposed to the sun in a dose-dependent manner. SCCs are likely derived from keratinocytes located near the skin surface. Aneuploidy is common in this type of cancer, as is the presence of p53 mutations. SCC may occur anywhere on the skin, although it may arise on the mucosal membranes of the mouth, nose, lips, throat, eyelids, lining of the breathing tubes, anus, cervix, etc.
Breast cancer is the most common form of malignant disease among women in Western countries. In the United States it is the most common cause of death among women between 40 and 55 years of age. The American Cancer Society predicts there will be over 183,000 new cases of invasive breast cancer in the year 2000 and over 40,000 deaths. The incidence of breast cancer is increasing, especially in older women, but the cause of this increase is unknown. Several types of breast cancer include ductal carcinoma in situ (DCIS), infiltrating (invasive) ductal carcinoma (IDC), lobular carcinoma in situ (LCIS) and infiltrating (invasive) lobular carcinoma (ILC). Risk factors linked to the disease include smoking, age, family history, race (Caucasians are more susceptible) and menses history (early onset of menses and menopause after age 50 increases the risk).
An even more deadly form of cancer is cancer of the brain. Every year over 100,000 people are diagnosed with a primary or metastatic brain tumor, which is the second leading cause of cancer death in children under age 15 and in cancer deaths of young adults up to age 34. The nature of brain tumors located at the center for thought, emotion and physical function renders them difficult to treat, and the cure rate is significantly lower than for most other types of cancer. Brain tumors originate from one cell and travel to other brain cells instead of traveling to other organs, as occurs with other types of cancer. Brain tumors can originate in the brain itself (such as astrocytoma, glioblastoma, oligodendroglioma, and ependymoma), in its coverings (meningiomas, pituitary tumors, pineal tumors), in the nerves at the base of the brain (acoustic neuromas, schwannomas), or from outside the brain (metastatic brain tumors). The most common forms of brain tumor are the malignant astrocytoma and glioblastoma multiforme. Brain tumors do not metastasize, so treatment is usually limited to the brain. Furthermore, true tumor margins do not exist, so complete removal by local therapy, such as surgery, radiation, heat, cold, etc. is prohibitive. Treatments for the whole brain are preferred, since brain tumor cells travel around the brain. Furthermore, brain tumors can be polyclonal containing multiple tumors in one malignant mass.
Although a host of treatments for cancer, including various forms of chemotherapy, have been developed there is still the need for treatments that target only the tumor-specific tissue and spare other normal tissue. Such treatments are the cause of side effects associated with existing cancer therapies.
B. Tumor Therapy
The lack of tumor-specificity remains a major problem for chemotherapy since the deleterious side effects prevent the delivery of doses of drugs that are required to eliminate tumors (Hoekman, 1999; Lowenthal and Eaton, 1996; Wada, 1999; Shin et al., 1998). For solid tumors, which comprise >90% of all human cancers, antibodies recognizing tumor-specific antigens provide little utility for drug delivery because the immunoconjugates are unable to penetrate tumor tissue (Dvorak et al., 1991; Shockley et al., 1991; Pietersz and McKenzie, 1992), resulting in a high level of cytotoxic drugs in blood and dose-limiting myelotoxicity.
In the past, antibodies recognizing tumor-specific antigens have been used to deliver cytotoxic drugs to tumors. However, such immunoconjugates have shown limited effectiveness towards solid tumors due to their inability to penetrate into the interior of tumor tissue.
Although some studies have shown the isolation of peptides that specifically localize to certain cell/tissue types, their localization to cancer specific cells has not been demonstrated, thereby limiting utility in cancer therapy. For example, Barry et al. (1996), describe the isolation of several peptides that show a greater affinity towards mouse fibroblasts as compared to random peptides. The authors utilized a fd phage-based random peptide display library for screening, but the absence of a subtraction step to eliminate nonspecifically interacting phages resulted in the isolation of peptides that bound to multiple cell types, including hepatoma, myoblast, and mastocytoma cells in addition to fibroblasts. Thus, the peptides isolated by Barry and colleagues lack specificity towards any particular tissues, thereby limiting their utility.
In another related study, Pasqualini et al. (1996) performed an in vivo screening of a random peptide display library and isolated several peptides that localize to mouse brain and kidney through interaction with endothelial cells specific to those organs. Although binding of the peptides to those organs appeared specific, since their interaction was diminished in the presence of specific competitors, these peptides target normal organs of non-human origin and do not demonstrate any utility for a cancer treatment.
Finally, studies by Arap et al. (1998) described attempts to block tumor growth indirectly by inhibiting angiogenesis. Through in vivo screening of a random peptide display library, peptides that localized to endothelial cells associated with human breast carcinoma xenografts were isolated. Some of the peptides contained Arg-Gly-Asp sequence, a motif that binds to a subset of integrins. By conjugating the cytotoxic drug doxorubicin to the peptide, a selective destruction of blood vessels associated with tumors was observed. This, in turn, resulted in the necrosis of the tumor and an increase in the survivability of the tumor-bearing mice. Pasqualini et al. (2000) have determined this peptide interacts with aminopeptidase N, although this protein is produced in multiple tissues.
Furthermore, the mechanism through which the doxorubicin-peptide conjugate enters cells has not been delineated. For example, the study does not address if the peptide is internalized. Also, as the peptide targets endothelial cells rather than tumor cells directly, it is not a direct tumor-specific agent.
Thus, there is a need for development of agents that can direct chemotherapeutics to specific human tumor cells, as opposed to tumor-associated cells such as tumor-associated endothelial cells, thereby preventing the delivery of these drugs to normal non-cancerous cells, which in turn will prevent generalized cell damage and the associated side effects.